家族序列相似性13A基因干扰对人气道上皮细胞凋亡和增殖的影响及与慢性阻塞性肺疾病患者小气道重塑的关系  

Effect of family with sequence similarity 13 member A gene interference on apoptosis and proliferation of human airway epithelial cells and its relationship with small airway remodeling in patients with chronic obstructive pulmonary disease

作　　者：朱金源[1] 马莉琼[2] 张锦[3] Zhu Jinyuan;Ma Liqiong;Zhang Jin(Department of Critical Care Medicine,General Hospital of Ningxia Medical University,Yinchuan 750004,China;Department of Pathology,General Hospital of Ningxia Medical University,Yinchuan 750004,China;Department of Respiratory and Critical Care Medicine,General Hospital of Ningxia Medical University,Yinchuan 750004,China)

机构地区：[1]宁夏医科大学总医院重症医学科,银川750004 [2]宁夏医科大学总医院病理科,银川750004 [3]宁夏医科大学总医院呼吸与危重症医学科,银川750004

出　　处：《中华医学杂志》2020年第32期2481-2487,共7页National Medical Journal of China

基　　金：国家自然科学基金(81760011);国家重点研发计划(2018YFC1313704);宁夏自然科学基金(2020AAC03378)。

摘　　要：目的探讨家族序列相似性13A(FAM13A)基因与慢性阻塞性肺疾病(简称慢阻肺)小气道重塑的关系,及干扰FAM13A基因表达对人气道上皮细胞(16HBE细胞)凋亡和增殖表型的影响。方法收集2018年1月至2020年1月宁夏医科大学总医院胸外科因肺部肿瘤或肺大泡行手术治疗的患者74例,根据肺功能和吸烟史分为4组:肺功能正常不吸烟组(正常组,23例)、肺功能正常吸烟组(吸烟组,24例)、慢阻肺不吸烟组(11例)和慢阻肺吸烟组(16例),采用免疫组化检测各组小气道FAM13A基因表达,并分析其与肺功能气流受限指标的相关性。设计FAM13A基因短片断干扰RNA(shRNA)片段,构建和包装FAM13A基因shRNA慢病毒载体,转染16HBE细胞,实时荧光定量PCR(qRT-PCR)和Western印迹法检测FAM13A基因表达水平,并筛选最佳shRNA序列。运用流式细胞技术检测细胞凋亡率和细胞增殖标志物Ki-67荧光强度。结果FAM13A主要表达于小气道上皮细胞的胞质,慢阻肺不吸烟组和慢阻肺吸烟组小气道上皮细胞FAM13A吸光度(A)值均高于正常组和吸烟组(0.365±0.026、0.412±0.053比0.113±0.018、0.105±0.009,均P<0.05),且其与肺功能气流受限指标第一秒用力呼气容积(FEV1)与用力肺活量(FVC)的比值(FEV1/FVC)及FEV1占预计值百分比(FEV1%pre)呈负相关(r=-0.48和r=-0.40,均P<0.05)。成功构建FAM13A基因shRNA慢病毒载体,并在16HBE细胞系上实现FAM13A干扰。转染16HBE细胞后,shRNA的靶标序列2(shRNA-target-2)的FAM13A表达量降低(均P<0.01);相比于阴性对照组(shRNA-NC),FAM13A shRNA组细胞凋亡率降低(P=0.023),且Ki-67荧光强度也降低(P=0.042)。结论FAM13A基因在慢阻肺小气道上皮细胞中表达增高,且与慢阻肺气流受限相关,FAM13A基因可能通过影响人气道上皮细胞凋亡和增殖而参与慢阻肺小气道重塑的过程。Objective To explore the relationship between family with sequence similarity 13 member A(FAM13A)gene and small airway remodeling in chronic obstructive pulmonary disease(COPD),and the effect of interference with FAM13A gene expression on the apoptosis and proliferation phenotype of human airway epithelial cells(16HBE).Methods From January 2018 to January 2020,74 patients in the Department of Thoracic Surgery of General Hospital of Ningxia Medical University were treated by surgery for lung tumors or pulmonary bullae.According to the lung function and smoking history,the 74 patients were divided into four groups:non-smoking group with normal lung function(normal group,23 patients),smoking group with normal lung function(smoking group,24 patients),non-smoking group with COPD(11 patients)and smoking group with COPD(16 patients).The expression of FAM13A in small airway of each group was detected by immunohistochemistry,and the correlation between FAM13A and the airflow restriction indexes by pulmonary function was analyzed.The shRNA fragment of FAM13A gene was designed,and the shRNA lentivirus vector of FAM13A gene was constructed and packaged.The expression level of FAM13A gene was detected by real-time fluorescent quantitative PCR(qRT-PCR)and Western blot,and the best shRNA sequence was screened.Flow cytometry was used to detect apoptosis rate and the fluorescence intensity of proliferation marker Ki-67 in 16HBE cells.Results FAM13A was mainly expressed in the cytoplasm of small airway epithelial cells.The levels of FAM13A absorbance(A)of small airway epithelial cells in non-smoking group and smoking group with COPD were higher than those in normal group and smoking group(0.365±0.026,0.412±0.053 to 0.113±0.018,0.105±0.009,all P<0.05),and they were negatively correlated with forced expiratory volume in 1s/forced vital capacity(FEV1/FVC)and FEV1%pre(r=-0.48 and r=-0.40,all P<0.05).The FAM13A shRNA lentiviral vector was successfully constructed,and FAM13A interference was successfully achieved in the 16HBE cell l

关 键 词：肺疾病 慢性阻塞性 气道重塑 细胞凋亡 细胞增殖 家族序列相似性13A基因 

分 类 号：R563.9[医药卫生—呼吸系统]